biomedicines Review Cannabis sativa: Interdisciplinary Strategies and Avenues for Medical and Commercial Progression Outside of CBD and THC Jackson M. J. Oultram 1, Joseph L. Pegler 1 , Timothy A. Bowser 2 , Luke J. Ney 3, Andrew L. Eamens 1 and Christopher P. L. Grof 1,2,* 1 Centre for Plant Science, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia; [email protected] (J.M.J.O.); [email protected] (J.L.P.); [email protected] (A.L.E.) 2 CannaPacific Pty Ltd., 109 Ocean Street, Dudley, NSW 2290, Australia; tim@cannapacific.com.au 3 School of Psychological Sciences, University of Tasmania, Hobart, TAS 7005, Australia; [email protected] * Correspondence: [email protected]; Tel.: +612-4921-5858 Abstract: Cannabis sativa (Cannabis) is one of the world’s most well-known, yet maligned plant species. However, significant recent research is starting to unveil the potential of Cannabis to pro- duce secondary compounds that may offer a suite of medical benefits, elevating this unique plant species from its illicit narcotic status into a genuine biopharmaceutical. This review summarises the lengthy history of Cannabis and details the molecular pathways that underpin the production of key secondary metabolites that may confer medical efficacy. We also provide an up-to-date summary of the molecular targets and potential of the relatively unknown minor compounds offered by the Cannabis plant. Furthermore, we detail the recent advances in plant science, as well as synthetic Citation: Oultram, J.M.J.; Pegler, J.L.; biology, and the pharmacology surrounding Cannabis. Given the relative infancy of Cannabis research, Bowser, T.A.; Ney, L.J.; Eamens, A.L.; Grof, C.P.L. Cannabis sativa: we go on to highlight the parallels to previous research conducted in another medically relevant and Interdisciplinary Strategies and versatile plant, Papaver somniferum (opium poppy), as an indicator of the possible future direction Avenues for Medical and Commercial of Cannabis plant biology. Overall, this review highlights the future directions of cannabis research Progression Outside of CBD and outside of the medical biology aspects of its well-characterised constituents and explores additional THC. Biomedicines 2021, 9, 234. avenues for the potential improvement of the medical potential of the Cannabis plant. https://doi.org/10.3390/ biomedicines9030234 Keywords: Cannabis sativa (Cannabis); cannabinoids; tetrahydrocannabinol (THC); cannabidiol (CBD); cannabinoid receptors (CB1 and CB2); Papaver somniferum (opium poppy); secondary metabolites Academic Editors: Pavel B. Drašar and Raffaele Capasso Received: 1 February 2021 1. Introduction Accepted: 23 February 2021 Published: 26 February 2021 Cannabis sativa (Cannabis) is arguably one of the world’s most versatile crops. While the genetic origin and evolution of Cannabis is a long-standing and heavily debated topic [1–4], Publisher’s Note: MDPI stays neutral in broad terms, today, Cannabis can be separated into two distinct categories, specifically with regard to jurisdictional claims in ‘hemp’ and ‘marijuana’. Much like other agricultural crop commodities, Cannabis has been published maps and institutional affil- domesticated and bred for thousands of years to produce phenotypic and/or chemotypic iations. traits of value to humans [2–5]. The chemotypic distinction between hemp and marijuana predominantly stems from the abundance of the principal psychoactive cannabinoid, D9- tetrahydrocannabinol (THC), present in the plant as the acidic form, D9-tetrahydrocannabinolic acid (THCA) [6]. To be considered hemp, Cannabis must possess a low percentage of THC relative to the total dry weight of flowers, with this low THC percentage varying from coun- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. try to country. In order to be legally cultivated as hemp, the cultivated plants must possess This article is an open access article less than 0.3% THC (w/w) in Canada [4,7] and China [8], whereas since 2001, the European distributed under the terms and Union determined that the THC content (w/w) of hemp must be below 0.2% [6]. conditions of the Creative Commons Hemp has traditionally been bred as a source for textile products due to the strong, Attribution (CC BY) license (https:// elongated bast fibres present in the phloem of the stem. More recently, the elevated cel- creativecommons.org/licenses/by/ lulosic content of hemp cell walls has garnered interest in the plant as a source for the 4.0/). Biomedicines 2021, 9, 234. https://doi.org/10.3390/biomedicines9030234 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 234 2 of 42 development of sustainable biofuel production [6]. Hempseed, and hempseed oil, have his- torically been utilised as a food source, with more contemporary research revealing their unique dietary value. In particular, the essential polyunsaturated fatty acids (PUFAs), linoleic acid (LA) and linolenic acid (LNA), comprise 50–70% and 15–25% of the total fatty acid content of hempseed, respectively; a 3:1 ratio promoted as nutritionally optimal [9–13]. PUFAs found in hempseed oil are incorporated into phospholipid bilayers and are integral to membrane fluidity and the maintenance of its permeability [14]. Moreover, the two pro- teins, edestin and albumin found in hempseed, contain rich amino acid profiles comparable to that of high-quality soybean and egg white [15]. Given the functions and importance of both fatty and amino acids, hempseed and hempseed oil may have some potential, albeit minor, for reducing the incidence of certain diseases, while in parallel conferring a range of health benefits [15–17]. Alternatively, marijuana has traditionally been bred for its recreational intoxication properties derived from the THCA-containing resin produced on the protruding secretory hair-like structures known as trichomes which are predom- inantly located on female reproductive parts of the Cannabis plant [18,19]. The sticky resin produced from these specialised epidermal glands is a rich mix of cannabinoid and non-cannabinoid constituents, numbering at least 104 and 441, respectively [20,21]. Most recently, two novel cannabinoids, namely D9-tetrahydrocannabiphorol (D9-THCP) and cannabidiphorol (CBDP), near identical in structure to THC and cannabidiol (CBD), respectively, were identified [22]. Notably, D9-THCP was demonstrated to possess higher cannabimimetic activity than THC, and its recent discovery is therefore postulated as a potential candidate cannabinoid responsible for variation in pharmacological properties ob- served in uncharacterised Cannabis varieties. This also identifies the likelihood of secondary metabolites present in Cannabis resin that remain to be discovered. In addition to possessing a range of phenotypic and chemotypic traits of interest to the textile, medicinal, food and energy industries as an agricultural crop, Cannabis is extremely versatile and hardy, hence the application of the colloquial term for this species, ‘weed’. The phenotypic flexibility of Cannabis provides it with the capacity to adapt and survive a range of abiotic and biotic insults, such as drought [23], heavy metal stress [24], high temperature [25], poor soil nutrient content [3], high plant density [26], and stem damage from the larva of Ostrinia nubilalis, the European corn borer [27]. Tolerance to a range of abiotic stress conditions is exemplified by the tap root of Cannabis which is able to adapt to highly variable edaphic conditions, either penetrating deep (greater than 2 metres) into dry soil, or developing an extensive lateral root network in response to its growth in soil that has a high moisture content [26]. Further, the widespread legalisation of medicinal application and recreational use of Cannabis is driving the growth of diverse research programs encompassing the broad scope, from plant breeding to clinical trials. In the United States of America (USA), for example, to date, 33 states have approved the medicinal use of Cannabis, while 14 states and territories have legalised the recreational use of marijuana by adults. At the federal level in the USA, however, Cannabis remains a ‘Schedule I Substance’. In direct contrast to the heavy legislation of Cannabis in the USA, its direct neighbour, Canada, legalised the use of Cannabis across the country in 2018 under the ‘Cannabis Act’[28]. As the legislative approval of Cannabis use increases worldwide, there will be an increasing need for interdisciplinary research to characterise secondary metabolites of interest and to increase the production of Cannabis to meet the demand for medicinal and recreational products. Currently, there exists an extant literature on the medical potential for the best charac- terised cannabinoids, THC and CBD [29–34]. Significantly less attention in medical research has been paid to the potential for the minor phytocannabinoids to treat illnesses, and there is still the need for methods to produce these cannabinoids cost-effectively for commercial production. In particular, the medical Cannabis industry faces significant challenges in multiple aspects of product development. For instance, THC is associated with multiple side effects, and furthermore, pharmaceutical-standard THC and CBD are expensive to produce. Due to these hurdles, many companies around the world which have attempted Biomedicines 2021, 9, 234 3 of 42 to capitalise on the increasing legality of Cannabis have been unsuccessful [35]. Therefore, here we review the current